The long-term goal of this project is to understand the mechanisms by which polyspermy is prevented in the tunicate Ascidia. In particular we are interested in the role(s) of membrane receptors (glycosides) in these processes. Ascidian sperm bind to these glycosides on the vitelline coat (VC) surface of the egg. Increasing numbers of sperm bind to the VC followed by a rapid decline. This decline in binding helps to insure that only a single sperm enters the egg. Sperm binding is inhibited by the Na+ dependent release of a specific glycosidase (N-acetyl-glucosaminidase, NAGase) that modifies the VC sperm receptors so that they no longer bind sperm. We believe that the NAGase is initially linked to the egg plasma membrane and is rapidly released following fertilization. Detergents and phospholipase C can free the enzyme from the unfertilized egg suggesting linkage to a membrane phospholipid. Our current model includes linkage of the NAGase to the phosphatidylinositol and the (fertilization induced?) Na+ dependent activation of a phospholipase that causes enzyme release. The specific aim of this present proposal is to continue to involve minority students in research investigating the block to polyspermy. Successful isolation of the phospholipase involved in NAGase release will allow us to determine if this enzyme is identical to the intracellular enzyme involved in Ca flux and second-messenger signaling.